WO2007032748A1 - Method for detecting dna methylation - Google Patents

Method for detecting dna methylation Download PDF

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WO2007032748A1
WO2007032748A1 PCT/SG2006/000271 SG2006000271W WO2007032748A1 WO 2007032748 A1 WO2007032748 A1 WO 2007032748A1 SG 2006000271 W SG2006000271 W SG 2006000271W WO 2007032748 A1 WO2007032748 A1 WO 2007032748A1
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dna
biotin
primer
primer set
methylation
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PCT/SG2006/000271
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French (fr)
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Masafumi Inoue
Kok Keong Lee
Hiroshi Ida
Yoshiaki Ito
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Agency For Science, Technology & Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a method for detecting methylation in DNA samples.
  • the invention relates to a method for detecting aberrant methylation of cytosine in CpG islands of tumour suppressor genes.
  • the invention also relates to the diagnosis of cancer.
  • Cancer is a major killer. Although in recent years the understanding of cancer has greatly improved, new and improved methods of early detection are still desirable.
  • DNA methylation is the addition of methyl groups ( -CH3) to cytosine (and sometimes adenine bases) in DNA. It occurs naturally and is thought to have a role in suppressing gene expression. As such, DNA methylation on tumour suppressor genes is often associated with cancer.
  • E- cadherin Genes such as E- cadherin, p16, p15 and RUNX3 are good examples for this methylation.
  • the p16 gene is a tumor suppressor gene and its methylation is associated with cancers. Since methylcytosine was found in genomic DNA, this epigenetic alteration has gained wide attention, especially because of its relation to gene silencing in disease leading to cancer.
  • methylcytosine is mainly found in cytosine-guanine (CpG) dinucleotides.
  • CpG cytosine-guanine
  • primers are used, which hybridize either only to a sequence that is formed by the bisulfite treatment of a DNA that is not methylated at the respective position, or, vice versa, primers, which only bind to a nucleic acid which has formed by the bisulfite treatment of a DNA that is methylated at the respective position.
  • amplified products can then be produced, whose detection in turn supplies hints of the presence of a methylated or unmethylated position in the sample, to which the primers bind.
  • the resulting products remain unclear whether this product is the specific products or not.
  • a method for detecting methylation in a DNA sample comprising the steps of:
  • the chemically converted DNA is amplified using a polymerase and a pair of oligonucleotide primers comprising an upper primer and a corresponding lower primer. More preferably, the upper primer carries the detectable label.
  • the agent chemically converts unmethylated cytosine residues in the DNA sample to uracil.
  • the oligonucleotide probe is bonded to a solid phase. More preferably, the solid phase a bead in a bead-array flow cytometry. Still more preferably, the reaction mix contains a plurality of beads, each bead has a different oligonucleotide probe to capture different target regions on the amplified DNA.
  • each bead has a different signature. More preferably that signature is a fluorescence wavelength such that each individual bead with a separate probe is distinguishable from other beads with different probes.
  • the label which is carried by the oligonucleotide primer is biotin and the further step of adding streptavidin wherein detecting the presence or absence of biotin is achieved by detecting a signal resulting from the interaction between biotin and streptavidin. More preferably, the added streptavidin is conjugated with a fluorescent label.
  • the amplification of the chemically converted DNA is carried out by means of a polymerase chain reaction. More preferably, the amplification comprises a multiplicity of oligonucleotide primers capable of simultaneously amplifying a plurality of genes of the DNA under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • the genes that are amplified may be selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLHl .
  • the agent that chemically converts the DNA is a bisulfite solution.
  • the polymerase that amplifies the chemically converted DNA is a heat- resistant DNA polymerase.
  • the methylation status of genes selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLHl in the DNA sample is determined in a single run.
  • a primer set comprising at least two primers capable of amplifying a portion of the sequence of p16 gene.
  • the primer set having the nucleotide sequence 5'-Biotin- GTTATTAGAGGGTGGGGCGGATCG-3' / 5'-AACGCCCCCGCCTCCAACAACG-3'.
  • a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the p16 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • a primer set comprising at least two primers capable of amplifying a portion of the sequence of RUNX3 gene.
  • the primer set having the nucleotide sequence 5'-(Biotin)- GGGATAGTTACGAGGGGCGGTCGTAC-3' / 5'- GACCGACGCGAACGCCTCCT -3'.
  • a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the RUNX3 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • a primer set comprising at least two primers capable of amplifying the promoter sequence of RASSF1A gene.
  • the primer set having the nucleotide sequence 5'-(Biotin)- TTTGCGAGAGCGCGTTTAG-3' / 5'-TAACAAACGCGAACCGAAC-3' or 5'- TTTGCGAGAGCGCGTTTAG-3' / 5'-(BiOtJn)-TAACAAACGCGAACCGAAC-3'.
  • a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the RASSF1A gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • a primer set comprising at least two primers capable of amplifying the promoter sequence of E- cadherin gene.
  • the primer set having the nucleotide sequence 5'-(Biotin)- AGGTTAGAGGGTTATCGCGTTTAT-3' / 5'-TACTTTACAATTCCGACGCCACT-3' or, 5'- AGGTTAGAGGGTTATCGCGTTTAT-3' / 5'-(Biotin)-TACTTTACAATTCCGACGCCACT- 3' or, 5'-(Biotin)-AGGTTAGAGGGTTATCGCGTTTATGC-3' / 5'-
  • a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the E-cadherin gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • a primer set comprising at least two primers capable of amplifying the promoter sequence of hMLH1 gene.
  • the primer set having the nucleotide sequence 5'-(Biotin)- ATTAATAGGAAGAGCGGATAGC-3' / 5'-CCTTCAACTATAACTTACGCCATC-3'.
  • a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the hMLH1 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of genes selected from RUNX3, p16, RASSF1A, E-cadherin and hMLH1 under a single set of reaction conditions in a multiplex polymerase chain reaction.
  • an oligonucleotide probe having a nucleotide sequence that hybridizes under stringent conditions to the complement of DNA sequences of RUNX3 and/or p16 and/or
  • the probes having the nucleotide sequences of:
  • CT 5'-H2N-C6-gggaggCgttgaagtTggggt-3';
  • TC 5'-H2N-C6-gggaggTgttgaagtCggggt-3';
  • CT 5'-H2N-C6-gggCggtgttttTggggttt-3';
  • a set of four probes are needed to detect the four methylation states in one reaction.
  • the four methylation states are: 1 all the cytosines are methylated; 2 only the first cytosine is methylated; 3 the second cytosine is methylated and; 4 where none of the cytosines are methylated. It is possible that more than 2 cytosine residues may be detected in which case there may be more that 4 DNA methylation states detected in one reaction.
  • the methylation states of more that one cancer suppressor gene can be measured in one reaction. This takes less time than traditional DNA methylation detection.
  • a fourteenth aspect of the invention there is provided a method of aiding assessment of a patient's risk of developing cancer, or progression of cancer, the method comprising the step of obtaining a sample containing nucleic acid from a patient and carrying out the method according to the first preferred aspect of the invention.
  • a method of diagnosing or assessing a patient's risk of developing cancer or other diseases by detecting DNA methylation in genes selected from the group RUNX3, p16, RASSF1A, E- cadherin and hMLH1, by carrying out the method according to the first preferred aspect of the invention.
  • a kit of parts comprising:
  • the kit further comprises a bisulfite agent.
  • the label which is carried by the oligonucleotide primer is biotin and the further step of adding streptavidin wherein detecting the presence or absence of biotin is achieved by detecting a signal resulting from the interaction between biotin and streptavidin. More preferably, the added streptavidin is conjugated with a fluorescent label.
  • the means of detecting the label is fluorescent flow cytometry. More preferably, fluorescence is detected after irradiation with two light sources the first allowing identification of the florescence signature of the bead and the specific probe indicating the possible DNA methylation state; and the second to detect hybridisation of a sample to that probe so the amount of sample in that methylation state can be calculated.
  • there are four methylation states 1 where all the cytosines are methylated, 2 where only the first cytosine is methylated, 3 where the second cytosine is methylated and 4 where none of the cytosines are methylated. It is possible that more than 2 cytosine residues may be detected in which case there may be more that 4 DNA methylation states detected in one reaction.
  • DNA methylation it is meant the addition of a methyl group to the carbon-5 position of cytosine residues, is the only common covalent modification of human DNA and occurs almost exclusively at cytosines that are followed immediately by a guanine (so- called CpG dinucleotides).
  • CpG dinucleotides a guanine
  • the bulk of the genome displays a clear depletion of CpG dinucleotides, and those that are present are nearly always methylated.
  • small stretches of DNA known as CpG islands, are comparatively rich in CpG nucleotides and are nearly always free of methylation.
  • CpG islands are frequently located within the promoter regions of human genes, and methylation within the islands has been shown to be associated with transcriptional inactivation of the corresponding gene. Alterations in DNA methylation might be pivotal in the development of most cancers. In recent years, it has become apparent that the pattern of DNA methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. Although cancers often exhibit clear reductions throughout their genomes in the levels of DNA methylation, this goes hand-in-hand with increased methylation at the CpG islands. Such changes in methylation have a central role in tumourigenesis; in particular, methylation of CpG islands has been shown to be important in transcriptional repression of numerous genes that function to prevent tumour growth or development. Studies of DNA methylation in cancer have thus opened up new opportunities for diagnosis, prognosis and ultimately treatment of human tumours.
  • the nucleic acid in the DNA sample is first chemically converted with bisulfite solution.
  • the solution is sodium bisulfate and it converts unmethylated cytosine to uracil. Methylated cytosine residues, on the other hand, would remain unchanged. Comparison of sodium bisulfate treated and untreated DNA reveal methylated cytosines.
  • the DNA samples may be obtained from a patient's cell lines, blood, sputum, stool, urine, serum, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from intestine, kidney, brain, heart, prostate, lung, eyes, breast or liver, histological slides and all possible combinations thereof.
  • a technique is also known as Methylation Specific PCR (MSP) which is a PCR technique for the study of DNA CpG methylation.
  • MSP Methylation Specific PCR
  • primers are used, which hybridize either only to a sequence that forms by the bisulfite treatment of a DNA which is unmethylated at the respective position, or, vice versa, primers which bind only to a nucleic acid which forms by the bisulfite treatment of a DNA which is methylated at the respective position.
  • the amplified DNA or amplicons can be produced accordingly with these primers.
  • Standard PCR has been described in US Patent Nos. 4,683,195; 4,800,195; and 4,965,188.
  • Primers are generally artificially synthesized, but they may be of recombinant origin or, in some applications, a mixture of both. They generally are used in pairs and comprise two nucleic acid sequences, one with a sense orientation (5'to 3') and one with an antisense (3'to 5'). They are generally used under optimized conditions for the purpose of identifying a specific gene or for diagnostic use.
  • oligonucleotide pairs may be used under less stringent or optimized conditions for identification and/or quantitation of closely related DNA or RNA sequences.
  • PCR-based techniques include (1) Inverse PCR, which is the first method to report successful acquisition of unknown sequences starting with primers based on a known region (Triglia, T. et al (1988) Nucleic Acids Res 16: 8186); (2) Capture PCR (Lagerstrom M. et al (1991) PCR Methods Applic 1 : 111-19) which is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and YAC DNA; (3) targeted gene walking (Parker J. D.
  • Primers developed according to the method of the present invention are added to a PCR assay in an amount effective for amplifying any target nucleic acid present in the sample. At least about 5 picomoles (moles) of each primer species may be used in a PCR reaction. It is known to use between 5 and 10 pmoles of primer per 50 ul PCR reaction, for optimum amplification (Perkin Elmer). At the minimum of 5 pmoles per species, a primer with 4 mixed base positions would have 5pm X 16 species, for a total of 80 pm. If both primers in the PCR reaction have 4 mixed positions, there would be a total of 2 X 80 pm, for a total of 160 pm of primers in a 50 microliter reaction. PCR and the resulting detection of PCR products are accomplished by methods known in the art.
  • primer it is meant to refer to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i. e., in the presence of four different nucleoside triphosphatase and an agent for polymerization (i. e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • a primer is preferably a single-stranded oligodeoxynucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 17 to about 23 nucleotides.
  • Primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template.
  • Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer (i. e., acting as a point of initiation of DNA synthesis).
  • primers may contain an additional nucleic acid sequence at the 5'end which does not hybridize to the target nucleic acid, but which facilitates cloning of the amplified product.
  • primers for use in processes such as PCR.
  • Various techniques for synthesizing oligonucleotide primers are well known in the art, including phosphotriester and phosphodiester synthesis methods.
  • the primers could at least 18-30, 40, 50, 100, 250 or 500 nucleotides in length.
  • DNA sequence or a portion or fragment thereof, entirely by synthetic chemistry using laboratory equipment familiar to the skilled artisans.
  • the source of information for producing the synthetic sequence may be derived from the known homologous sequence from closely related organisms. After synthesis, the nucleic acid sequence can be used alone or joined with another known sequence and inserted into one of the many available DNA vectors and their respective host cells using techniques well known in the art.
  • synthetic chemistry may be used to introduce specific mutations into the nucleic acid sequence. Alternatively, a portion of sequence in which a mutation is desired can be synthesized and recombined with a portion of an existing genomic or recombinant sequence.
  • amplification it is meant to refer to an increase in the amount of the desired nucleic acid molecule present in a sample.
  • amplicon or “amplified DNA” it is meant to refer to a small DNA fragment which is the product of PCR or LCR; a piece of DNA that has been synthesized using amplification techniques.
  • each bead has a different signature. More preferably that signature is a fluorescence wavelength such that each individual bead with a separate probe is distinguishable from other beads with different probes.
  • hybridization it is meant to refer to the formation of a duplex structure by two single- stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between substantially complementary nucleic acid strands that contain minor regions of mismatch. Conditions under which only fully complementary nucleic acid strands will hybridize are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions.
  • nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair concentration of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e. g., Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1985), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wilty- Interscience, John Wiley and Sons, N. Y. (1987 updated quarterly) (each incorporated herein by reference).
  • hybridizes under stringent conditions it is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other.
  • the conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C , followed by one or more washes in 0.2. times. SSC, 0.1% SDS at 50-65°C. Examples of moderate to low stringency hybridization conditions are well known in the art.
  • DNA typically contains a polynucleotide composed of the 4 "natural" bases: A (adenine), T (thymine), C (cytosine), and G (guanine).
  • A adenine
  • T thymine
  • C cytosine
  • G guanine
  • the hydrogen bonding (or base pairing) among these nucleotides creates the double-stranded structure of a DNA molecule.
  • An A-containing residue base pairs to a T-containing residue through the formation of two hydrogen bonds; a G-containing residue base pairs to a C-containing residue through the formation of three hydrogen bonds.
  • target region and “target nucleic acid” it refers to a region of a nucleic acid which is to be amplified, detected, or otherwise analyzed.
  • Target nucleic acid capable of being amplified through use of the primers developed by the methods of the present invention includes any prokaryotic or eukaryotic nucleic acid (DNA or RNA), which can include any plant, animal, bacterial, viral, fungal, etc., nucleic acids for which at least one nucleic acid sequence is known.
  • the sequence to which a primer or probe hybridizes can be referred to as a "target sequence”.
  • probes it is meant to refer to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences in a particular cell or tissue or test sample. In the present invention, they are used to hybridise to complementary regions of the DNA that is amplified. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single-or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, bead- array flow cytometry or ELISA-like technologies.
  • Oligonucleotides such as primers and probes, can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al.,
  • Bead-array is an array of DNA probes on beads in a capillary with an order determined by the probe species and hybridization is performed by the reciprocal flow of the sample.
  • Beads of 5.6- ⁇ m diameter are lined in a determined order in a capillary. Each bead can have a different DNA probe to capture different DNA targets. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 ⁇ l) into the capillary with reciprocal flow.
  • DNA micro-array technology may be used.
  • DNA micro-array is an array of DNA probes that are respectively synthesized using either photolithographic techniques or liquid-spotting methods. It has been used for various applications including gene expression profiling and mutation detection. Although it is a very powerful and attractive device, it is very expensive and a practical fabrication method for producing a cost- effective device is still required. In addition, it is impossible to rearrange any of the probes in the array in accordance with changes in the analysis target. This requirement seems to be overcome by micro-spheres having DNA probes.
  • the bead-array is designed so as to decrease the volume of reaction space to ⁇ 0.1 ⁇ l to enable fast hybridization.
  • the bead size was determined to be 5.6 ⁇ m in diameter.
  • 1 to 100 beads or more may be placed in a capillary, which occupy ⁇ 1-cm length in the capillary.
  • the capillary is connected to a sample reservoir, buffer reservoir, waste reservoir and a syringe pump with capillary tubing.
  • the device is placed in a thermal chamber.
  • a sample solution including target DNAs tagged with the detectable label is introduced into the bead-array from the sample reservoir and continuously flowed reciprocally by operating the syringe pump.
  • the beads are washed with buffer solutions introduced from the buffer reservoirs.
  • the bead-array is illuminated by a light source and the fluorescence intensity of each bead is measured with a CCD camera.
  • detecting a signal it includes any optically detectable chromophoric or fluorescent signals that are associated with the functioning of the particular model system used.
  • signals will generally be produced by products of the enzyme's catalytic action, e.g., on a chromogenic or fluorogenic substrate.
  • binding systems e.g., receptor ligand interactions
  • signals will typically involve the association of a labelled ligand with the receptor, or vice versa.
  • labels it includes labels that are commonly detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful nucleic acid labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • Labelling agents include e.g., monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any of a variety of known methods, including spectrophotometric or optical tracking of radioactive or fluorescent markers, or other methods which track a molecule based upon size, charge or affinity.
  • a detectable moiety can be of any material having a detectable physical or chemical property.
  • detectable labels have been well-developed in the field of gel electrophoresis, column chromatography, solid substrates, spectroscopic techniques, and the like, and in general, labels useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical thermal, or chemical means.
  • Useful labels in the present invention include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, 32P or 33P), enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker products or as in an ELISA), nucleic acid intercalators (e.g., ethidium bromide) and calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3H, 1251, 35S, 14C, 32P or 33P
  • Fluorescent labels are particularly preferred labels.
  • Preferred labels are typically characterized by one or more of the following: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labelling.
  • Fluorescent moieties which are incorporated into the labels of the invention, are generally are known, including 1- and 2-aminonaphthalene, p,p'-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone imines, anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bis- benzoxazole, bis-p-oxazolyl benzene, 1 ,2-benzophenazin, retinol, bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3- chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin
  • Individual fluorescent compounds which have functionalities for linking to an element desirably detected in an apparatus or assay of the invention, or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthaIene; N-phenyl 2-amino- 6-suIfonatonaphthalene; 4-acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid; pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl-N-methyl-2- aminoaphthalene-6-sulfonate; ethidium bromide; stebrine; auromine-0,2-(9'- anthroyl)palmitate; dans
  • fluorescent tags are commercially available from SIGMA chemical company (Saint Louis, Mo.), Molecular Probes, R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica- Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.) as well as other commercial sources known to one skilled.
  • fluorescent labels absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound label may differ from the unbound label. Therefore, when referring to the various wavelength ranges and characteristics of the labels, it is intended to indicate the labels as employed and not the label which is unconjugated and characterized in an arbitrary solvent.
  • Fluorescent labels are one preferred class of detectable labels, in part because by irradiating a fluorescent label with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events.
  • Detectable signal may also be provided by chemiluminescent and bioluminescent sources.
  • Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor.
  • a diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions. One family of compounds is 2,3-dihydro-1,4-phthalazinedione. The most popular compound is luminol, which is a 5- amino compound.
  • Chemiluminescent analogs include para-dimethylamnino and -methoxy substituents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds are also known and available, including -N-alkyl acridinum esters (basic H.sub.2 O.sub.2) and dioxetanes. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence.
  • the label may coupled directly or indirectly to a molecule to be detected (a product, substrate, enzyme, or the like) according to methods well known in the art.
  • a molecule to be detected a product, substrate, enzyme, or the like
  • Non radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with labeled, anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody. Labels can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label
  • it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as digital cameras, charge coupled devices (CCDs) or photomultipliers and phototubes, and the like. Fluorescent labels and detection techniques, particularly microscopy and spectroscopy are preferred. Similarly, enzymatic labels are detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple calorimetric labels are often detected simply by observing the colour associated with the label. For example, conjugated gold often appears pink, while various conjugated beads appear the colour of the bead.
  • the status of methylation pattern of the gene is then determined by detection of the label fluorescent intensity in a bead-array flow cytometric assay.
  • the results obtained were compared with the results by both gel based MSP and bisulfite direct DNA sequencing.
  • the present method provides detection of aberrant DNA methylation in tumour suppressor genes in a high sensitive and yet specific manner.
  • the present method may be used to detect methylation of the genes RUNX3, p16, RASSF1A, E-cadherin and hMLHL Therefore, the following primers and probes may be used by the present method:
  • H2N denotes amino modifier for cross linking to the bead.
  • C6 denotes carbon spacer
  • RASSF1A S-Probe1CC 5'-H2N-C6-gggaggCgttgaagtCggggt-3'(Bead #17)
  • RASSF1A S-Probe1CT 5'-H2N-C6-gggaggCgttgaagtTggggt-3' (Bead #18)
  • RASSF1A S-Probe1TC 5'-H2N-C6-gggaggTgttgaagtCggggt-3' Bead #19)
  • RASSF1 A S-Probe1TT 5'-H2N-C6-gggaggTgttgaagtTggggt-3' (Bead #20)
  • Ecad S-Probe 1CC 5'-H2N-C6-gggCggtgttttCggggttt-3' (Bead #73)
  • Ecad S-Probe 1CT 5'-H2N-C6-gggCggtgttttTggggttt-3' (Bead #74)
  • Ecad S-Probe 1TC 5'-H2N-C6-gggTggtgtttttCggggttt-3' (Bead #75)
  • Ecad S-Probe 1TT 5'-H2N-C6-gggTggtgttttTggggttt-3' (Bead #76)
  • a method of aiding assessment of a patient's risk of developing cancer, or progression of cancer comprising the step of obtaining a sample containing nucleic acid from a patient and carrying out the method in accordance with the first preferred aspect of the invention. This may be carried out by detecting DNA methylation in any or all of the genes selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLH1.
  • the CpG islands in p16 and RUNX3 genes are as follows:
  • the cytosines in the CpG dinucleotides are generally unmethylated.
  • sequences (particularly promoter sequences) of genes associated with certain cancers or inherited diseases such as RUNX3, p16, RASSF1A, E-cadherin and hMLH1 , these cytosines can be methylated. Therefore, the method of the present invention can diagnose/assess a patient's risk of developing cancer or other diseases by detecting DNA methylation in those genes.
  • the primer/primers and probe/probes according to the invention may be provided as part of a kit, e. g. in a suitable container such as a vial in which the contents are protected from the external environment.
  • the kit may include instructions for use of the nucleic acid, e. g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample such as the hybridisation technique described herein.
  • a kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc.
  • the nucleic acid may be labelled.
  • a kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself.
  • reagents for performance of the method such as means for providing the test sample itself.
  • the label carried on the primer is biotin
  • a further reagent including streptavidin may be added to detect the nucleic acid of interest.
  • the streptavidin may be conjugated with a fluorescent label.
  • the kit includes one or more primers for the determination of the presence of methylation of those genes.
  • Preferred primers are described as above.
  • Test samples were created by 10-fold serially diluting bisulfite-modified completely methylated DNA with bisulfite-modified completely unmethylated DNA and labeled as 100, 10, 1, 0.1 , 0.01, and 0.001. Sample 100 is thus 100% of completely methylated DNA and no completely unmethylated DNA. Sample 10 is 10% of completely methylated DNA and 90% completely unmethylated DNA and so forth.
  • the promoter region of p16 sequence was obtained from NCBI Locus AB060808. The sequence was then created by converting the original genomic sequence to bisulfite DNA sequence based on the principle in which all CpG's cytosines are methylated and thus remain as C ( Figure 1).
  • Primer set The methylation specific primers were selected.
  • the sense and antisense methylation specific primers for this experiment are as follows: Upper primer: IMCB B-p16M3-5S (699U24, C:2) : 5'-Biotin- GTTATTAGAGGGTGGGGCGGATCG-3' Lower primer.
  • IMCB p16M3-3AS (868L22, G:3): 5'- AACGCCCCCGCCTCCAACAACG-3'
  • the expected amplicon sizes created by this primer set is 191 bp.
  • PCR was performed using the QlAGEN HotStrart Taq PCR kit (catalog no. 210210).
  • PCR products made by the above primer set were analyzed by conventional agarose gel electrophoresis and ethidium bromide staining.
  • Luminex(R) 100 (TM) system TX, USA was used.
  • H2N denotes amino modifier for cross linking to the bead.
  • C6 denotes carbon spacer -
  • xMAP(Tm)-Carboxylated Microspheres were obtained from the supplier (Luminex(R), TX, USA). They are internally labeled with fluorescent dyes and contain surface carboxyl groups for covalent attachment of ligands (or biomolecules).
  • the -COOH on the surface on the bead and -NH2 of the oligo probe are cross linked by Carbodiimide coupling, according to the manufactures' protocol (Luminex(R), TX, USA). There are 100 kinds of beads are commercially available.
  • the number 151, 152, 153 and 154 were selected and bound with GG, AG, GA and AA probe respectively for p16 probes in this experiment.
  • 5 x 10(6) beads were reacted with 0.2 nmol of each probe by 0.04%(w/v) EDC [1-ethyl- 3(3-dimethylaminopropyl) carbodiimide HCI (Pierce #77149) in 0.1 M MES buffer [2-(N- Morpholino)ethanesulfonic acid Hydrate (Sigma M2933)] at pH 4.5.
  • the beads were counted by Luminex(R) 100 after the coupling step.
  • Step 1 Sample preparation
  • 5ul of the PCR product was hybridized in the presence of 5ul of the Beads mix and 40ul of 1x TMAC hybridization solution [3 M TMA (Sigma Cat. T-3411), 0.1% Sodium lauroyl Sarcosinate (Sigma Cat. L-5125), 50mM Tris-HCI, pH 8.0 (Sigma Cat. T3038), 4mM EDTA, pH 8.0 (Gibco Cat.15575-038)].
  • the mixture was denatured at 95°C for 10 minutes and incubated at 60°C for 40 minutes.
  • washing buffer [1x Phosphate Buffer Saline, 0.01% Tween 20 (Sigma Cat. P1379)] was added to each well at room temperature and centrifuged at 2000g for 10 minutes. The supernatant was decanted and another 100ul of washing buffer was added followed by incubation at 52°C for 10 minutes. The mixture was centrifuged at 2000g for 10 minutes and the supernatant was decanted.
  • Step 4 Signal production 70 ul of washing buffer (1x Phosphate Buffer Saline, 0.01% Tween 20) containing X1/500 Streptavidin, R-phycoerythrin conjugate (Molecular Probes, Cat. S866) was added and mixed by repeat pipetting. The mixture was incubated at 52°C for 5 minutes.
  • the fluorescent signal was read by Luminex 100.
  • Figures 6 to 8 shows the sensitivity and specificity of Beads-MSP in low and high stringent conditions in a methylation specific PCR using p16.
  • the detection of DNA methylation using bead-array platform is more specific robust than the conventional agarose electrophoresis and ethidium bromide.
  • Met-DNA detection using bead-array platform DNA is treated with bisulfite as discussed in Example 1.
  • RASSF1A Set 1 (Upper primer is biotinylated)
  • RASSF1A S-Probe1CC 5'-H2N-C6-gggaggCgttgaagtCggggt-3'(Bead #17)
  • RASSF1A S-Probe1CT 5'-H2N-C6-gggaggCgttgaagtTggggt-3' (Bead #18)
  • RASSF1A S-Probe1TC 5'-H2N-C6-gggaggTgttgaagtCggggt-3' Bead #19)
  • RASSF1A S-Probe1TT 5'-H2N-C6-gggaggTgttgaagtTggggt-3' (Bead #20)
  • Ecad S-Probe 1CC 5'-H2N-C6-gggCggtgttttCggggttt-3' (Bead #73)
  • Ecad S-Probe 1CT 5'-H2N-C6-gggCggtgttttTggggttt-3' (Bead #74)
  • Ecad S-Probe 1TC 5'-H2N-C6-gggTggtgttttCggggttt-3' (Bead #75)
  • Ecad S-Probe 1 TT 5'-H2N-C6-gggTggtgtttTggggttt-3' (Bead #76)
  • Multiplex PCR was performed using the QIAGEN Multiplex PCR kit (catalog no. 206143). 5ul of DNA, RUNX3 upper primer and lower primer at a final concentration of 0.1 uM and 0.05uM, p16 upper and lower primer at a final concentration of 0.1 uM and 0.1uM, RASSF1A upper and lower primer at a final concentration of 0.2uM and 0.1uM, E-cadherin upper and lower primer at a final concentration of 0.2uM and 0.1 uM, hMLH1 upper and lower primer at a final concentration of 0.2uM and 0.1 uM, and GAPDH upper and lower primer at a final concentration of 0.05uM and 0.025uM in 50uI final volume, on a Stratagene Robocycler 40 (La JoIIa, California) by using the following steps: initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 45 s, annealing at 58°
  • Example 1 Bead coupling for the probe is described in Example 1. The hybridisation and detection techniques are also carried out as described in Example 1.
  • Figure 9 shows graphically the results of simultaneous detection of DNA methylation in tumour suppressor genes by a bead-based flow cytometric assay.

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Abstract

The invention relates to methods for detecting methylatiÌn in DNA samples, particular aberrant methylation of cytosine in CpG islands of tumour suppressor genes. The method involves: a) treating the DNA sample with bisulfite solution to convert unmethylated cytosine to uracil; b) amplifying the DNA using oligonucleotide primers carrying a detectable label; c) hybridizing the amplified DNA to an oligonucleotide probe; d) determining the methylation status of the DNA sample through detecting the presence or absence of label of the hybridized amplified DNA. Primers and probes for carrying out the method are also disclosed.

Description

METHOD FOR DETECTING DNA METHYLATION
Field of invention
The invention relates to a method for detecting methylation in DNA samples. In particular, but not exclusively, the invention relates to a method for detecting aberrant methylation of cytosine in CpG islands of tumour suppressor genes. The invention also relates to the diagnosis of cancer.
Background of the Invention
Cancer is a major killer. Although in recent years the understanding of cancer has greatly improved, new and improved methods of early detection are still desirable.
People are more likely to survive cancer if the disease is diagnosed at an early stage of development, since treatment at that time is more likely to be successful. Early detection depends upon availability of high-quality methods. Such methods are also useful for determining patient prognosis, selecting therapy, monitoring response to therapy and selecting patients for additional therapy. Consequently, there is a need for cancer diagnostic methods that are specific, accurate, minimally invasive, technically simple and inexpensive.
DNA methylation is the addition of methyl groups ( -CH3) to cytosine (and sometimes adenine bases) in DNA. It occurs naturally and is thought to have a role in suppressing gene expression. As such, DNA methylation on tumour suppressor genes is often associated with cancer.
Cytosines located 5' to the guanines, called CpG dinucleotides or islands, are present in the regulatory regions of many genes. In normal cells, the cytosines in the CpG dinucleotides are generally unmethylated. However, as mentioned above, in the promoter sequences of genes associated with certain cancers or inherited diseases, these cytosines can be methylated. This suggests that methylation may be involved in the transcription control of a cancerous cell in humans. Methylation recruits a variety of transcriptional repressors, including histone deacetylases and other proteins that cause chromosome condensation and silencing (Schubeler et al., 2000; reviewed by Bestor, 1998). Genes such as E- cadherin, p16, p15 and RUNX3 are good examples for this methylation. The p16 gene is a tumor suppressor gene and its methylation is associated with cancers. Since methylcytosine was found in genomic DNA, this epigenetic alteration has gained wide attention, especially because of its relation to gene silencing in disease leading to cancer.
In animals, methylcytosine is mainly found in cytosine-guanine (CpG) dinucleotides. The presence of 5-methylcytosine in the promoter of specific genes alters the binding of transcriptional factors and other proteins to DNA in plants and animals, block transcription and cause gene silencing.
Thus, methylation of C residues in genomic DNA plays a key role in the regulation of gene expression.
There are a wide range of techniques available for the study of the occurrence and localization of methylcytosine in the genome (Fraga et al., 2002). One of the most commonly used techniques is methylation specific PCR(Herman J G, Graff J R,
Myohanen S, Nelkin B D, Baylin S B. (1996), Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. September 3; 93
(18): 9821-6). For this method, primers are used, which hybridize either only to a sequence that is formed by the bisulfite treatment of a DNA that is not methylated at the respective position, or, vice versa, primers, which only bind to a nucleic acid which has formed by the bisulfite treatment of a DNA that is methylated at the respective position.
With these primers, amplified products can then be produced, whose detection in turn supplies hints of the presence of a methylated or unmethylated position in the sample, to which the primers bind. However the resulting products remain unclear whether this product is the specific products or not.
Another method is bisulfite DNA sequencing. While this method reveals wide range of methylation status in the gene, it is a time consuming procedure. Summary of the Invention
In accordance with a first preferred aspect of the invention, there is provided a method for detecting methylation in a DNA sample, the method comprising the steps of:
(a) contacting the DNA sample with an agent that chemically converts the DNA;
(b) amplifying the chemically converted DNA using a polymerase and at least one oligonucleotide primer carrying a detectable label; and (c) hybridizing the amplified DNA to at least one oligonucleotide probe; wherein the methylation status of the DNA sample is determined by detecting the presence or absence of the label as a result of whether the amplified DNA hybridizes to the probe.
Preferably, the chemically converted DNA is amplified using a polymerase and a pair of oligonucleotide primers comprising an upper primer and a corresponding lower primer. More preferably, the upper primer carries the detectable label.
Preferably, the agent chemically converts unmethylated cytosine residues in the DNA sample to uracil.
Preferably, the oligonucleotide probe is bonded to a solid phase. More preferably, the solid phase a bead in a bead-array flow cytometry. Still more preferably, the reaction mix contains a plurality of beads, each bead has a different oligonucleotide probe to capture different target regions on the amplified DNA.
Preferably each bead has a different signature. More preferably that signature is a fluorescence wavelength such that each individual bead with a separate probe is distinguishable from other beads with different probes.
Preferably, the label which is carried by the oligonucleotide primer is biotin and the further step of adding streptavidin wherein detecting the presence or absence of biotin is achieved by detecting a signal resulting from the interaction between biotin and streptavidin. More preferably, the added streptavidin is conjugated with a fluorescent label.
Preferably, the amplification of the chemically converted DNA is carried out by means of a polymerase chain reaction. More preferably, the amplification comprises a multiplicity of oligonucleotide primers capable of simultaneously amplifying a plurality of genes of the DNA under a single set of reaction conditions in a multiplex polymerase chain reaction. The genes that are amplified may be selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLHl .
Preferably, the agent that chemically converts the DNA is a bisulfite solution.
Preferably, the polymerase that amplifies the chemically converted DNA is a heat- resistant DNA polymerase.
Preferably, the methylation status of genes selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLHl in the DNA sample is determined in a single run.
In accordance with a second aspect of the invention, there is provided a primer set comprising at least two primers capable of amplifying a portion of the sequence of p16 gene. Preferably, the primer set having the nucleotide sequence 5'-Biotin- GTTATTAGAGGGTGGGGCGGATCG-3' / 5'-AACGCCCCCGCCTCCAACAACG-3'.
In accordance with a third aspect of the invention, there is provided a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the p16 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
In accordance with a fourth aspect of the invention, there is provided a primer set comprising at least two primers capable of amplifying a portion of the sequence of RUNX3 gene. Preferably, the primer set having the nucleotide sequence 5'-(Biotin)- GGGATAGTTACGAGGGGCGGTCGTAC-3' / 5'- GACCGACGCGAACGCCTCCT -3'. In accordance with a fifth aspect of the invention, there is provided a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the RUNX3 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
In accordance with a sixth aspect of the invention, there is provided a primer set comprising at least two primers capable of amplifying the promoter sequence of RASSF1A gene. Preferably, the primer set having the nucleotide sequence 5'-(Biotin)- TTTGCGAGAGCGCGTTTAG-3' / 5'-TAACAAACGCGAACCGAAC-3' or 5'- TTTGCGAGAGCGCGTTTAG-3' / 5'-(BiOtJn)-TAACAAACGCGAACCGAAC-3'.
In accordance with a seventh aspect of the invention, there is provided a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the RASSF1A gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
In accordance with a eighth aspect of the invention, there is provided a primer set comprising at least two primers capable of amplifying the promoter sequence of E- cadherin gene. Preferably, the primer set having the nucleotide sequence 5'-(Biotin)- AGGTTAGAGGGTTATCGCGTTTAT-3' / 5'-TACTTTACAATTCCGACGCCACT-3' or, 5'- AGGTTAGAGGGTTATCGCGTTTAT-3' / 5'-(Biotin)-TACTTTACAATTCCGACGCCACT- 3' or, 5'-(Biotin)-AGGTTAGAGGGTTATCGCGTTTATGC-3' / 5'-
TACTTTACAATTCCGACGCCACT-3' or, 5'-AGGTTAGAGGGTTATCGCGTTTATGC-3' / 5'-(Biotin)-TACTTTACAATTCCGACGCCACT-3'.
In accordance with a ninth aspect of the invention, there is provided a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the E-cadherin gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
In accordance with a tenth aspect of the invention, there is provided a primer set comprising at least two primers capable of amplifying the promoter sequence of hMLH1 gene. Preferably, the primer set having the nucleotide sequence 5'-(Biotin)- ATTAATAGGAAGAGCGGATAGC-3' / 5'-CCTTCAACTATAACTTACGCCATC-3'. In accordance with a eleventh aspect of the invention, there is provided a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the hMLH1 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
In accordance with a twelfth aspect of the invention, there is provided a primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of genes selected from RUNX3, p16, RASSF1A, E-cadherin and hMLH1 under a single set of reaction conditions in a multiplex polymerase chain reaction.
In accordance with a thirteenth aspect of the invention, there is provided an oligonucleotide probe having a nucleotide sequence that hybridizes under stringent conditions to the complement of DNA sequences of RUNX3 and/or p16 and/or
RASSF1A and/or E-cadherin and/or hMLHL Preferably, the probes having the nucleotide sequences of:
GG : 5'-H2NC6-tctcccctctccGcaaccGcc -3';
AG : 5'-H2NC6-tctcccctctccAcaaccGcc -3';
GA : 5'-H2NC6-tctcccctctccGcaaccAcc -3';
AA : 5'-H2N-C6-tctccccctctccAcaaccAcc -3;'
GG: 5'-H2N-C6-ctacccGactaatcccGcatc -3';
GA: 5'-H2N-C6-ctacccGactaatcccAcatc -3';
AG: 5'-H2N-C6-ctacccAactaatcccGcatc -3';
AA: 5'-H2N-C6-ctacccAactaatcccAcatc -3';
GG: 5'-H2N-C6-accccGacttcaacGcctcc -3';
AG: 5'-H2N-C6-accccAacttcaacGcctcc -3';
GA: 5'-H2N-C6-accccGacttcaacAcctcc -3';
AA: 5'-H2N-C6-accccAacttcaacAcctccc -3';
GG: 5'-H2N-C6-accccGaaaacaccGccccc -3';
AG: 5'-H2N-C6-accccAaaaacaccGccccc -3';
GA: 5'-H2N-C6-accccGaaaacaccAccccc -3';
AA: 5'-H2N-C6-accccAaaaacaccAccccc -3';
GG: 5'-H2N-C6-taaaacGactactacccGctacc -3';
AG: 5'-H2N-C6-taaaacAactactacccGctacc -3'; GA: 5'-H2N-C6-taaaacGactactacccActacc -3';
AA: 5'-H2N-C6-taaaacAactactacccActacc -3';
CC: 5'-H2N-C6-gggaggCgttgaagtCggggt-3';
CT: 5'-H2N-C6-gggaggCgttgaagtTggggt-3';
TC: 5'-H2N-C6-gggaggTgttgaagtCggggt-3';
TT: 5'-H2N-C6-gggaggTgttgaagtTggggt-3';
CC: 5'-H2N-C6-gggCggtgttttCggggttt-3';
CT: 5'-H2N-C6-gggCggtgttttTggggttt-3';
TC: 5'-H2N-C6-gggTggtgttttCggggttt-3';
TT: 5'-H2N-C6-gggTggtgttttTggggttt-3'; or
5'-H2N-C6-ccatatacccaaaaaaaccacacc-3'.
Preferably, a set of four probes are needed to detect the four methylation states in one reaction. Preferably the four methylation states are: 1 all the cytosines are methylated; 2 only the first cytosine is methylated; 3 the second cytosine is methylated and; 4 where none of the cytosines are methylated. It is possible that more than 2 cytosine residues may be detected in which case there may be more that 4 DNA methylation states detected in one reaction.
Preferably, the methylation states of more that one cancer suppressor gene can be measured in one reaction. This takes less time than traditional DNA methylation detection.
In accordance with a fourteenth aspect of the invention, there is provided a method of aiding assessment of a patient's risk of developing cancer, or progression of cancer, the method comprising the step of obtaining a sample containing nucleic acid from a patient and carrying out the method according to the first preferred aspect of the invention.
In accordance with a fifteenth aspect of the invention, there is provided a method of diagnosing or assessing a patient's risk of developing cancer or other diseases by detecting DNA methylation in genes selected from the group RUNX3, p16, RASSF1A, E- cadherin and hMLH1, by carrying out the method according to the first preferred aspect of the invention. In accordance with a sixteenth aspect of the invention, there is provided a kit of parts comprising:
(a) one or more primers according to the present invention, wherein the primer or primers carry a detectable label; (b) one or more probe conjugated beads according to the present invention;
(c) means for detecting the label; and
(d) directions for performing the method according to the first preferred aspect of the invention.
Preferably, the kit further comprises a bisulfite agent.
Preferably, the label which is carried by the oligonucleotide primer is biotin and the further step of adding streptavidin wherein detecting the presence or absence of biotin is achieved by detecting a signal resulting from the interaction between biotin and streptavidin. More preferably, the added streptavidin is conjugated with a fluorescent label.
Preferably the means of detecting the label is fluorescent flow cytometry. More preferably, fluorescence is detected after irradiation with two light sources the first allowing identification of the florescence signature of the bead and the specific probe indicating the possible DNA methylation state; and the second to detect hybridisation of a sample to that probe so the amount of sample in that methylation state can be calculated. Preferably, there are four methylation states, 1 where all the cytosines are methylated, 2 where only the first cytosine is methylated, 3 where the second cytosine is methylated and 4 where none of the cytosines are methylated. It is possible that more than 2 cytosine residues may be detected in which case there may be more that 4 DNA methylation states detected in one reaction.
Detailed Description of the Preferred Embodiments
A high throughput yet simple method has been developed to detect aberrant methylated DNA using a bead-array flow cytometric assay. By "DNA methylation" it is meant the addition of a methyl group to the carbon-5 position of cytosine residues, is the only common covalent modification of human DNA and occurs almost exclusively at cytosines that are followed immediately by a guanine (so- called CpG dinucleotides). The bulk of the genome displays a clear depletion of CpG dinucleotides, and those that are present are nearly always methylated. By contrast, small stretches of DNA, known as CpG islands, are comparatively rich in CpG nucleotides and are nearly always free of methylation. These CpG islands are frequently located within the promoter regions of human genes, and methylation within the islands has been shown to be associated with transcriptional inactivation of the corresponding gene. Alterations in DNA methylation might be pivotal in the development of most cancers. In recent years, it has become apparent that the pattern of DNA methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. Although cancers often exhibit clear reductions throughout their genomes in the levels of DNA methylation, this goes hand-in-hand with increased methylation at the CpG islands. Such changes in methylation have a central role in tumourigenesis; in particular, methylation of CpG islands has been shown to be important in transcriptional repression of numerous genes that function to prevent tumour growth or development. Studies of DNA methylation in cancer have thus opened up new opportunities for diagnosis, prognosis and ultimately treatment of human tumours.
The nucleic acid in the DNA sample is first chemically converted with bisulfite solution. Preferably, the solution is sodium bisulfate and it converts unmethylated cytosine to uracil. Methylated cytosine residues, on the other hand, would remain unchanged. Comparison of sodium bisulfate treated and untreated DNA reveal methylated cytosines.
The DNA samples may be obtained from a patient's cell lines, blood, sputum, stool, urine, serum, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from intestine, kidney, brain, heart, prostate, lung, eyes, breast or liver, histological slides and all possible combinations thereof.
The treated (chemically converted) DNA derived from methylated DNA and unmethylated DNA and then subjected to a polymerase chain reaction with biotinylated methylation specific primers. Such a technique is also known as Methylation Specific PCR (MSP) which is a PCR technique for the study of DNA CpG methylation. For the MSP experiments, primers are used, which hybridize either only to a sequence that forms by the bisulfite treatment of a DNA which is unmethylated at the respective position, or, vice versa, primers which bind only to a nucleic acid which forms by the bisulfite treatment of a DNA which is methylated at the respective position. The amplified DNA or amplicons can be produced accordingly with these primers.
Standard PCR has been described in US Patent Nos. 4,683,195; 4,800,195; and 4,965,188. Primers are generally artificially synthesized, but they may be of recombinant origin or, in some applications, a mixture of both. They generally are used in pairs and comprise two nucleic acid sequences, one with a sense orientation (5'to 3') and one with an antisense (3'to 5'). They are generally used under optimized conditions for the purpose of identifying a specific gene or for diagnostic use. In addition, the same two oligonucleotide pairs, a pair of "nested" oligonucleotides, or a pool of degenerate oligonucleotides may be used under less stringent or optimized conditions for identification and/or quantitation of closely related DNA or RNA sequences.
Other useful PCR-based techniques include (1) Inverse PCR, which is the first method to report successful acquisition of unknown sequences starting with primers based on a known region (Triglia, T. et al (1988) Nucleic Acids Res 16: 8186); (2) Capture PCR (Lagerstrom M. et al (1991) PCR Methods Applic 1 : 111-19) which is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and YAC DNA; (3) targeted gene walking (Parker J. D. et al (1991 ; Nucleic Acids Res 19: 3055-60) which is a method for targeted gene walking which permits retrieval of unknown sequence; and (4) Capillary Electrophoresis which is a new method for analyzing either the size or the nucleic acid sequence of PCR products.
Primers developed according to the method of the present invention are added to a PCR assay in an amount effective for amplifying any target nucleic acid present in the sample. At least about 5 picomoles (moles) of each primer species may be used in a PCR reaction. It is known to use between 5 and 10 pmoles of primer per 50 ul PCR reaction, for optimum amplification (Perkin Elmer). At the minimum of 5 pmoles per species, a primer with 4 mixed base positions would have 5pm X 16 species, for a total of 80 pm. If both primers in the PCR reaction have 4 mixed positions, there would be a total of 2 X 80 pm, for a total of 160 pm of primers in a 50 microliter reaction. PCR and the resulting detection of PCR products are accomplished by methods known in the art.
It is particularly preferred to conduct the amplifications of several different fragments with more than 2 different primers in one reaction vessel and thus to carry out the amplification steps as a multiplex PCR. It is generally particularly preferred to conduct the amplifications as a polymerase chain reaction.
By "primer" it is meant to refer to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i. e., in the presence of four different nucleoside triphosphatase and an agent for polymerization (i. e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligodeoxynucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 17 to about 23 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer (i. e., acting as a point of initiation of DNA synthesis). For example, primers may contain an additional nucleic acid sequence at the 5'end which does not hybridize to the target nucleic acid, but which facilitates cloning of the amplified product.
Those skilled in the art are well versed in the design of primers for use in processes such as PCR. Various techniques for synthesizing oligonucleotide primers are well known in the art, including phosphotriester and phosphodiester synthesis methods. The primers could at least 18-30, 40, 50, 100, 250 or 500 nucleotides in length.
It is also possible to produce a DNA sequence, or a portion or fragment thereof, entirely by synthetic chemistry using laboratory equipment familiar to the skilled artisans. The source of information for producing the synthetic sequence may be derived from the known homologous sequence from closely related organisms. After synthesis, the nucleic acid sequence can be used alone or joined with another known sequence and inserted into one of the many available DNA vectors and their respective host cells using techniques well known in the art. Moreover, synthetic chemistry may be used to introduce specific mutations into the nucleic acid sequence. Alternatively, a portion of sequence in which a mutation is desired can be synthesized and recombined with a portion of an existing genomic or recombinant sequence.
By "amplification" it is meant to refer to an increase in the amount of the desired nucleic acid molecule present in a sample. By "amplicon" or "amplified DNA" it is meant to refer to a small DNA fragment which is the product of PCR or LCR; a piece of DNA that has been synthesized using amplification techniques.
The amplicon or amplified DNA products were then hybridized with specific probes conjugated to fluorescent beads. Preferably each bead has a different signature. More preferably that signature is a fluorescence wavelength such that each individual bead with a separate probe is distinguishable from other beads with different probes.
By "hybridization" it is meant to refer to the formation of a duplex structure by two single- stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between substantially complementary nucleic acid strands that contain minor regions of mismatch. Conditions under which only fully complementary nucleic acid strands will hybridize are referred to as "stringent hybridization conditions" or "sequence-specific hybridization conditions". Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair concentration of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e. g., Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1985), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wilty- Interscience, John Wiley and Sons, N. Y. (1987 updated quarterly) (each incorporated herein by reference). By "hybridizes under stringent conditions" it is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C , followed by one or more washes in 0.2. times. SSC, 0.1% SDS at 50-65°C. Examples of moderate to low stringency hybridization conditions are well known in the art.
DNA typically contains a polynucleotide composed of the 4 "natural" bases: A (adenine), T (thymine), C (cytosine), and G (guanine). The hydrogen bonding (or base pairing) among these nucleotides creates the double-stranded structure of a DNA molecule. An A-containing residue base pairs to a T-containing residue through the formation of two hydrogen bonds; a G-containing residue base pairs to a C-containing residue through the formation of three hydrogen bonds.
By "target region" and "target nucleic acid" it refers to a region of a nucleic acid which is to be amplified, detected, or otherwise analyzed. Target nucleic acid capable of being amplified through use of the primers developed by the methods of the present invention includes any prokaryotic or eukaryotic nucleic acid (DNA or RNA), which can include any plant, animal, bacterial, viral, fungal, etc., nucleic acids for which at least one nucleic acid sequence is known. The sequence to which a primer or probe hybridizes can be referred to as a "target sequence".
By "probes" it is meant to refer to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences in a particular cell or tissue or test sample. In the present invention, they are used to hybridise to complementary regions of the DNA that is amplified. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single-or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, bead- array flow cytometry or ELISA-like technologies.
Oligonucleotides, such as primers and probes, can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al.,
Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth.
Enzymol. 68 : 109-151 (1979) ; the diethylphosphoramidite method of Beaucage et al.,
Tetrahedron Lett. 22: 1859-1862 (1981); and the solid support method of U. S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods is provided in Goodchild, Bioconjugate Chemistry 1 (3) : 165-187 (1990), incorporated herein by reference.
Bead-array, is an array of DNA probes on beads in a capillary with an order determined by the probe species and hybridization is performed by the reciprocal flow of the sample.
Beads of 5.6-μm diameter are lined in a determined order in a capillary. Each bead can have a different DNA probe to capture different DNA targets. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 μl) into the capillary with reciprocal flow.
Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer.
Also, DNA micro-array technology may be used. DNA micro-array is an array of DNA probes that are respectively synthesized using either photolithographic techniques or liquid-spotting methods. It has been used for various applications including gene expression profiling and mutation detection. Although it is a very powerful and attractive device, it is very expensive and a practical fabrication method for producing a cost- effective device is still required. In addition, it is impossible to rearrange any of the probes in the array in accordance with changes in the analysis target. This requirement seems to be overcome by micro-spheres having DNA probes. The combination of color- coded microbeads and a flow cytometer, massive parallel signature sequence, which uses microbeads for cDNA cloning and parallel sequencing reactions, and fixed microbeads mounted on the terminal wells of optical fibers have been reported.
The bead-array is designed so as to decrease the volume of reaction space to <0.1 μl to enable fast hybridization. In the present invention, the bead size was determined to be 5.6 μm in diameter. 1 to 100 beads or more may be placed in a capillary, which occupy <1-cm length in the capillary. The capillary is connected to a sample reservoir, buffer reservoir, waste reservoir and a syringe pump with capillary tubing. The device is placed in a thermal chamber. A sample solution including target DNAs tagged with the detectable label is introduced into the bead-array from the sample reservoir and continuously flowed reciprocally by operating the syringe pump. After the hybridization is completed, the beads are washed with buffer solutions introduced from the buffer reservoirs. Finally, the bead-array is illuminated by a light source and the fluorescence intensity of each bead is measured with a CCD camera.
By "detecting a signal" it includes any optically detectable chromophoric or fluorescent signals that are associated with the functioning of the particular model system used. For enzyme systems, such signals will generally be produced by products of the enzyme's catalytic action, e.g., on a chromogenic or fluorogenic substrate. For binding systems, e.g., receptor ligand interactions, signals will typically involve the association of a labelled ligand with the receptor, or vice versa.
A wide variety of other detectable signals and labels can also be used in the assays and apparatuses of the invention. In addition to the chromogenic and fluorogenic labels described above, radioactive decay, electron density, changes in pH, solvent viscosity, temperature and salt concentration are also conveniently measured. By "labels", it includes labels that are commonly detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful nucleic acid labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. A wide variety of labels suitable for labelling biological components are known and are reported extensively in both the scientific and patent literature, and are generally applicable to the present invention for the labelling of biological components. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Labelling agents optionally include e.g., monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any of a variety of known methods, including spectrophotometric or optical tracking of radioactive or fluorescent markers, or other methods which track a molecule based upon size, charge or affinity. A detectable moiety can be of any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of gel electrophoresis, column chromatography, solid substrates, spectroscopic techniques, and the like, and in general, labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical thermal, or chemical means. Useful labels in the present invention include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, 32P or 33P), enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker products or as in an ELISA), nucleic acid intercalators (e.g., ethidium bromide) and calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
Fluorescent labels are particularly preferred labels. Preferred labels are typically characterized by one or more of the following: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labelling.
Fluorescent moieties, which are incorporated into the labels of the invention, are generally are known, including 1- and 2-aminonaphthalene, p,p'-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone imines, anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bis- benzoxazole, bis-p-oxazolyl benzene, 1 ,2-benzophenazin, retinol, bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3- chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin, porphyrins, triarylmethanes and flavin. Individual fluorescent compounds which have functionalities for linking to an element desirably detected in an apparatus or assay of the invention, or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthaIene; N-phenyl 2-amino- 6-suIfonatonaphthalene; 4-acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid; pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl-N-methyl-2- aminoaphthalene-6-sulfonate; ethidium bromide; stebrine; auromine-0,2-(9'- anthroyl)palmitate; dansyl phosphatidylethanolamine; N,N'-dioctadecyl oxacarbocyanine: N,N'-dihezyl oxacarbocyanine; merocyanine, 4-(3'pyrenyl)stearate; d-3-aminodesoxy- equilenin; 12-(9'-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene; 2,2'(vinylene- p-phenylene)bisbenzoxazole; p-bis(2-(4-methyl-5-phenyl-oxazolyl))benzene; 6- dimethylamino-1 ,2-benzophenazin; retinol; bis(3'-aminopyridinium) 1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin; chiorotetracycline; N-(7-dimethylamino- 4-methyl-2-oxo-3-chromenyl)maleimide; N-(p-(2-benzimidazolyl)-pheny)maleimide; N-(4- fluoranthyl)maleimide; bis(homovanillic acid); resazarin; 4-chloro-7-nitro-2,1 ,3- benzooxadiazole; merocyanine 540; resorufin; rose bengal; and 2,4-diphenyl-3(2H)- furanone. Many fluorescent tags are commercially available from SIGMA chemical company (Saint Louis, Mo.), Molecular Probes, R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica- Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.) as well as other commercial sources known to one skilled.
Desirably, fluorescent labels absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound label may differ from the unbound label. Therefore, when referring to the various wavelength ranges and characteristics of the labels, it is intended to indicate the labels as employed and not the label which is unconjugated and characterized in an arbitrary solvent.
Fluorescent labels are one preferred class of detectable labels, in part because by irradiating a fluorescent label with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events. Detectable signal may also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions. One family of compounds is 2,3-dihydro-1,4-phthalazinedione. The most popular compound is luminol, which is a 5- amino compound. Other members of the family include the 5-amino-6,7,8-trimethoxy- and the dimethylaminolca[ca]benz analog. These compounds can be made to luminesce with alkaline hydrogen peroxide or calcium hypochlorite and base. Another family of
' compounds is the 2,4,5-triphenylimidazoles, with lophine as the common name for the parent product. Chemiluminescent analogs include para-dimethylamnino and -methoxy substituents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds are also known and available, including -N-alkyl acridinum esters (basic H.sub.2 O.sub.2) and dioxetanes. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence.
The label may coupled directly or indirectly to a molecule to be detected (a product, substrate, enzyme, or the like) according to methods well known in the art. As indicated above, a wide variety of labels are used, with the choice of label depending on the sensitivity required, ease of conjugation of the compound, stability requirements^ available instrumentation, and disposal provisions. Non radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to a polymer. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with labeled, anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody. Labels can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol. Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as digital cameras, charge coupled devices (CCDs) or photomultipliers and phototubes, and the like. Fluorescent labels and detection techniques, particularly microscopy and spectroscopy are preferred. Similarly, enzymatic labels are detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple calorimetric labels are often detected simply by observing the colour associated with the label. For example, conjugated gold often appears pink, while various conjugated beads appear the colour of the bead.
The status of methylation pattern of the gene is then determined by detection of the label fluorescent intensity in a bead-array flow cytometric assay. The results obtained were compared with the results by both gel based MSP and bisulfite direct DNA sequencing. The present method provides detection of aberrant DNA methylation in tumour suppressor genes in a high sensitive and yet specific manner.
The present method may be used to detect methylation of the genes RUNX3, p16, RASSF1A, E-cadherin and hMLHL Therefore, the following primers and probes may be used by the present method:
A. p16 p16 Primer set
Upper primer: IMCB B-p16M3-5S (699U24, C:2) : 5'-Biotin-
GTTATTAGAGGGTGGGGCGGATCG-3'
Lower primer: IMCB p16M3-3AS (868L22, G:3): 5'- AACGCCCCCGCCTCCAACAACG-3' p16 Capturing Probes
For 5-C-C-3' case (2 methylations positive case)= p16 Probe2++ GG : 5'-H2NC6- tctcccctctccGcaaccGcc -3'
For 5-C-T-3' case (1 methylation positive case) = p16 Probe2++ AG : 5'-H2NC6- tctcccctctccAcaaccGcc -3'
For 5T-C-3 case (1 methylation positive case) = p16 Probe2++ GA : 5'-H2NC6- tctcccctctccGcaaccAcc -3'
For 5T-T-3 case (methylation negative case) = p16 Probe2++ AA : 5-H2N-C6- tctccccctctccAcaaccAcc -3' H2N denotes amino modifier for cross linking to the bead. C6 denotes carbon spacer -
CCCCCC- between probe and bead.
A. p16 (for multiplex PCR) p16 Primer set Upper: 5'-(Biotin)-TATTAGAGGGTGGGGCGGATC-3'
Lower: 5'-GAACCGCGACCGTAACCAA-3' p16 Probes
For 5'-C-C-3' case (2 methylations positive case)= p16 Probe2++ GG : 5'-H2NC6- tctcccctctccGcaaccGcc -3' For 5-C-T-3' case (1 methylation positive case) = p16 Probe2++ AG : 5'-H2NC6- tctcccctctccAcaaccGcc -3'
For 5T-C-3 case (1 methylation positive case) = p16 Probe2++ GA : 5'-H2NC6- tctcccctctccGcaaccAcc -3'
For 5T-T-3 case (methylation negative case) = p16 Probe2++ AA : 5'-H2N-C6- tctccccctctccAcaaccAcc -3'
H2N denotes amino modifier for cross linking to the bead. C6 denotes carbon spacer
CCCCCC- between probe and bead.
B. RUNX3 RUNX3 Primer set
Upper: 5'-(Biotin)-GGGATAGTTACGAGGGGCGGTCGTAC-3'
Lower: 5'- GACCGACGCGAACGCCTCCT -3'
RUNX3 Capturing Probes
GG: 5'-H2N-C6-ctacccGactaatcccGcatc -3'
GA: 5'-H2N-C6-ctacccGactaatcccAcatc -3'
AG: 5'-H2N-C6-ctacccAactaatcccGcatc -3'
AA: 5'-H2N-C6-ctacccAactaatcccAcatc -3'
C. RASSF1A
Set 1 (Upper primer is biotinylated)
RASSF1A Primer set
Upper: 5'-(Biotin)-TTTGCGAGAGCGCGTTTAG-3'
Lower: 5'-TAACAAACGCGAACCGAAC-3'
RASSF1A Capturing Probes
GG: 5'-H2N-C6-accccGacttcaacGcctcc -3' (Bead #17)
AG: 5'-H2N-C6-accccAacttcaacGcctcc -3' (Bead #18)
GA: 5'-H2N-C6-accccGacttcaacAcctcc -3' (Bead #19)
AA: 5'-H2N-C6-accccAacttcaacAcctccc -3' (Bead #20)
Set 2 (Lower primer is biotinylated)
RASSF1A Primer set
Upper: 5'-TTTGCGAGAGCGCGTTTAG-3'
Lower 5'-(biotin)-TAACAAACGCGAACCGAAC-3'
RASSF1A Capturing Probes
RASSF1A S-Probe1CC: 5'-H2N-C6-gggaggCgttgaagtCggggt-3'(Bead #17)
RASSF1A S-Probe1CT: 5'-H2N-C6-gggaggCgttgaagtTggggt-3' (Bead #18)
RASSF1A S-Probe1TC: 5'-H2N-C6-gggaggTgttgaagtCggggt-3' Bead #19)
RASSF1 A S-Probe1TT: 5'-H2N-C6-gggaggTgttgaagtTggggt-3' (Bead #20)
D. E-cadherin Set 1 (Upper primer is biotinylated)
E-cadherin Primer set
Upper: 5'-(Biotin)-AGGTTAGAGGGTTATCGCGTTTATGC-3' or 5'-(Biotin)-
AGGTTAGAGGGTTATCGCGTTTAT-3'
Lower: 5'-TACTTTACAATTCCGACGCCACT-3'
E-cadherin Capturing Probes
GG: 5'-H2N-C6-accccGaaaacaccGccccc -3' (Bead #73)
AG: 5'-H2N-C6-accccAaaaacaccGccccc -3' (Bead #74)
GA: 5'-H2N-C6-accccGaaaacaccAccccc -3' (Bead #75)
AA: 5'-H2N-C6-accccAaaaacaccAccccc -3' (Bead #76)
Set 2 (Lower primer is biotinylated)
E-cadherin Primer set
Upper: 5'- AGGTTAGAGGGTTATCGCGTTTATGC-3' or 5'-(Biotin)- AGGTTAGAGGGTTATCGCGTTTAT-3' Lower: 5'-(biotin)-TACTTTACAATTCCGACGCCACT-3' E-cadherin Capturing Probes
Ecad S-Probe 1CC: 5'-H2N-C6-gggCggtgttttCggggttt-3' (Bead #73) Ecad S-Probe 1CT: 5'-H2N-C6-gggCggtgttttTggggttt-3' (Bead #74) Ecad S-Probe 1TC: 5'-H2N-C6-gggTggtgttttCggggttt-3' (Bead #75) Ecad S-Probe 1TT: 5'-H2N-C6-gggTggtgttttTggggttt-3' (Bead #76)
E. hMLH1 hMLM Primer set
Upper: 5'-(Biotin)-ATTAATAGGAAGAGCGGATAGC-3'
Lower: 5'-CCTTCAACTATAACTTACGCCATC-3'
hMLH1 Probes
GG: 5'-H2N-C6-taaaacGactactacccGctacc -3' AG: 5'-H2N-C6-taaaacAactactacccGctacc -3' GA: 5'-H2N-C6-taaaacGactactacccActacc -3' AA: 5'-H2N-C6-taaaacAactactacccActacc -3'
F. GAPDH, human
(This is for DNA control.) hGAPDH Primer set
Upper: 5'-Biotin-GTTTTGGGGAGGTAATTAGGAT-3'
Lower: 5'-CTCACCCCAACCTTCTCCATAATA-3' hGAPDH probe
5'-H2N-C6-ccatatacccaaaaaaaccacacc-3'
(only 1 probe)
A number of the methods for making probes corresponding to a target sequence for such purposes are known in the art.
In another embodiment of the invention, there is provided a method of aiding assessment of a patient's risk of developing cancer, or progression of cancer, the method comprising the step of obtaining a sample containing nucleic acid from a patient and carrying out the method in accordance with the first preferred aspect of the invention. This may be carried out by detecting DNA methylation in any or all of the genes selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLH1.
The CpG islands in p16 and RUNX3 genes are as follows:
481 TGTTTTCGGT TGGTGTTTTC GGGGGAGATT TAATTTGGGG CGATT TTAGG GGTGTTATAT 541 TCGTTAaGTG TTCGGAGTTA ATAGTATTTT TTTCGAGTAT TCGTTTACGG CGTTTTTTTG 601 TTTGGAAAGA TATCGCGGTT TTTTTAGAGG ATTTGAGGGΛ TAGGGTCGGA GGGGGTTTTT 661 TCGTTAGTAT CGGAGGAAGA AAGAGGAGGG GTTGGTTGGT TATTAGAGGG TGGGGCGGAT 721 CGCGTGCGTT CGGCGGTTGC QGAGAGGGGG AGAGTAGGTA GGGGGCGGCG GGGAGTAGTA 781 TGGAGTCGGC GGGGGGGAGT AGTATGGAGT TTSCGGTTGA TTGGTTGGTT ACGGTCGCGG 841 TTCGGGGTCG GGTAGAGGAG GTGCGGGCGT TGTTGGAGGC GGGGGGGTTG TTTAACGTAT 901 CGAATAGTTA CGGTCGGAGG TCGATTTAGG TGGGTAGAGG GTTTGTAGCG GGAGTAGGGG 961 ATGGCGGGCG ATTTTGGAGG ACGAAGTTTG TAGGGGAATT GGAATTAGGT AGCGTTTCGA 1021 TTTTTCGGAA AAAGGGGAGG TTTTTTGGGG AGTTTTTAGA AGGGGTTTGT AATTATAGAT 1081 TTTTTTTTGG CGACGTTTTG GGGGTTTGGG AAGTTAAGGA AGAGGAATGA GGAGTTACGC
RUNX3
29661 GGGAGTTACG ATTCGAGAGA GGGCGGTAAG GGCGTTTTTC GTGGGATTCG GACGTTTTAA 29661 GTAAATTTTT AGTATTTGTT TCGGGTTTTT AGAGWSTGG GGGGTTTTGG GTTGTGGTAT 29721 TGGGGTTTTT TTCGCGGGGT GGCGTTTTTC GTTTTTTTTG GTTGGGCGGT TTTCGGTAGG 29781 TTT0GTTTTT TTTCGCGAAC GTTATCGAGG TGTTGGCGAT GGGGGTTTCG TCGATTGGTT 29S41 GTGCGACGCG TCGTTTCGTT AGTTTCGTTT CGCGGGTTTC GGGGGTATTA ATTTCGCGCG 29901 GGCGGTCGCG GTTT0GTTAT TTGATTTTGG AGGATTTGTT TTGGGGTTGC GGTCGCGGAG 29961 TCGGGGCGGT CGCGGGCGAG TTTCGGGGCG GGAGGCGGCG GTAGCGGTAT AGTTTCGCGC 30021 GGGTTTCGTC GCGGTTTAGG TAGTCGGGAT AGTTACGAGG GGCGGTCGTA CGCGGGGTCG 30081 CGCGTCGAGG ATGCGGGATT AGTCGGGTAG GTTGCGGGCG GTCGTCGGGT TAGCGAGGTT 30141 TCGTAGCGGG CGGGTTTTGG CGAGTAGTGG TCGGGCGTCG TTTTTTGCGT TTTGAGGTTC 30201 GGGTTTCGTC GTTTTTGTTT TTTCGTTTTT CGCGGTAGCG GCGGTCGAGG AGGCGTTCGC
In normal cells, the cytosines in the CpG dinucleotides are generally unmethylated. However, in sequences (particularly promoter sequences) of genes associated with certain cancers or inherited diseases such as RUNX3, p16, RASSF1A, E-cadherin and hMLH1 , these cytosines can be methylated. Therefore, the method of the present invention can diagnose/assess a patient's risk of developing cancer or other diseases by detecting DNA methylation in those genes.
The primer/primers and probe/probes according to the invention may be provided as part of a kit, e. g. in a suitable container such as a vial in which the contents are protected from the external environment. The kit may include instructions for use of the nucleic acid, e. g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample such as the hybridisation technique described herein. A kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc. The nucleic acid may be labelled. A kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself. For example, if the label carried on the primer is biotin, a further reagent including streptavidin may be added to detect the nucleic acid of interest. The streptavidin may be conjugated with a fluorescent label.
In preferred embodiments of the invention the kit includes one or more primers for the determination of the presence of methylation of those genes. Preferred primers are described as above.
The invention will now be described in more detail by reference to the following Examples:
Example 1
Materials and Methods
Control DNA
Completely methylated and completely unmethylated DNA were purchased from Chemicon International, CA, USA (CpGenomeTM Universal Methylated DNA set, Cat. #S7821 and CpGenomeTM Universal Unmethylated DNA Set, Cat.# S7822) All CpG islands in completely methylated DNA are methylated. All CpG islands in unmethylated DNA are completely unmethylated.
Bisufite conversion of DNA samples
All DNAs were bisulfite converted prior to the methylation specific PCR. Bisulfite conversion of DNA samples was carried out essentially as described (Herman et al, 1996) and was based on the principle that treatment of DNA with bisulfite would result in the conversion of unmethylated cytosine residues into uracil. Methylated cytosine residues, on the other hand, would remain unchanged. Thus, the DNA sequences of methylated and unmethylated genomic regions following bisulfite conversion would be different and distinguishable by sequence-specific PCR primers. Bisulfite conversion was carried out using reagents provided in a CpGenome DNA Modification Kit (Chemicon, International, CA, USA, Cat. #S7820). One mg of DNA was treated with sodium bisulfite, followed by sulfonation, deamination & desulphonation according to the manufacturers' recommendations.
Preparation of test samples
Test samples were created by 10-fold serially diluting bisulfite-modified completely methylated DNA with bisulfite-modified completely unmethylated DNA and labeled as 100, 10, 1, 0.1 , 0.01, and 0.001. Sample 100 is thus 100% of completely methylated DNA and no completely unmethylated DNA. Sample 10 is 10% of completely methylated DNA and 90% completely unmethylated DNA and so forth.
p16 DNA Methylation Specific PCR
The promoter region of p16 sequence was obtained from NCBI Locus AB060808. The sequence was then created by converting the original genomic sequence to bisulfite DNA sequence based on the principle in which all CpG's cytosines are methylated and thus remain as C (Figure 1).
Primer set The methylation specific primers were selected. The sense and antisense methylation specific primers for this experiment are as follows: Upper primer: IMCB B-p16M3-5S (699U24, C:2) : 5'-Biotin- GTTATTAGAGGGTGGGGCGGATCG-3' Lower primer. IMCB p16M3-3AS (868L22, G:3): 5'- AACGCCCCCGCCTCCAACAACG-3' The expected amplicon sizes created by this primer set is 191 bp.
PCR condition
PCR was performed using the QlAGEN HotStrart Taq PCR kit (catalog no. 210210). In brief, 5ul of DNA, upper primer at a final concentration of 0.2 uM and lower primer at a final concentration of 0.1 uM on a Stratagene Robocycler 40 (La JoIIa1 California) by using the following steps: initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 42 s, annealing at 68°C for 58 s, and extension at 72°C for 26 s, and a final extension at 72°C for 5 min.
Detection by gel electrophoresis (for comparison with the results obtained using a method according to the present invention, i.e. detection by bead-array platform)
The PCR products made by the above primer set were analyzed by conventional agarose gel electrophoresis and ethidium bromide staining.
Detection by bead-array platform
Platform
In this experiment Luminex(R) 100 (TM) system (TX, USA) was used.
Probe and beads
(a) Probes The following sequence found in the amplicon was selected for the subsequent hybridization assay: 5'ggCggttgCggagagggggaga-3' (C denotes methyltion site on the sequence.)
Based on this sequence, four probes are designed as such: For 5-C-C-3' case (2 methylations positive case)= p16 Probe2++ GG : 5'-H2NC6- tctcccctctccGcaaccGcc -3'
For 5-C-T-3' case (1 methylation positive case) = p16 Probe2++ AG : 5'-H2NC6- tctcccctctccAcaaccGcc -3'
For 5T-C-3 case (1 methylation positive case) = p16 Probe2++ GA : 5'-H2NC6- tctcccctctccGcaaccAcc -3'
For 5T-T-3 case (methylation negative case) = p16 Probe2++ AA : 5'-H2N-C6- tctccccctctccAcaaccAcc -3'
H2N denotes amino modifier for cross linking to the bead. C6 denotes carbon spacer -
CCCCCC- between probe and bead.
(b) Beads
The beads
5.6 micron polystyrene microspheres which are internally dyed with red and infrared fluorophores called xMAP(Tm)-Carboxylated Microspheres were obtained from the supplier (Luminex(R), TX, USA). They are internally labeled with fluorescent dyes and contain surface carboxyl groups for covalent attachment of ligands (or biomolecules).
Beads coupling
The -COOH on the surface on the bead and -NH2 of the oligo probe are cross linked by Carbodiimide coupling, according to the manufactures' protocol (Luminex(R), TX, USA). There are 100 kinds of beads are commercially available.
This offers the potential to assay up to 100 different analytes in a single sample.
The number 151, 152, 153 and 154 were selected and bound with GG, AG, GA and AA probe respectively for p16 probes in this experiment. 5 x 10(6) beads were reacted with 0.2 nmol of each probe by 0.04%(w/v) EDC [1-ethyl- 3(3-dimethylaminopropyl) carbodiimide HCI (Pierce #77149) in 0.1 M MES buffer [2-(N- Morpholino)ethanesulfonic acid Hydrate (Sigma M2933)] at pH 4.5. The beads were counted by Luminex(R) 100 after the coupling step.
Detection
Detection was done as follows. The flow diagram of the procedure is shown in Figure 2.
Step 1 : Sample preparation
5ul of the PCR product was hybridized in the presence of 5ul of the Beads mix and 40ul of 1x TMAC hybridization solution [3 M TMA (Sigma Cat. T-3411), 0.1% Sodium lauroyl Sarcosinate (Sigma Cat. L-5125), 50mM Tris-HCI, pH 8.0 (Sigma Cat. T3038), 4mM EDTA, pH 8.0 (Gibco Cat.15575-038)].
Step 2: Denaturation and Hybridization
The mixture was denatured at 95°C for 10 minutes and incubated at 60°C for 40 minutes.
Step 3: Post hybridization washing
After hybridization, the mixture was centrifuged at 2000g for 10 minutes and the supernatant was decanted. 100ul of washing buffer [1x Phosphate Buffer Saline, 0.01% Tween 20 (Sigma Cat. P1379)] was added to each well at room temperature and centrifuged at 2000g for 10 minutes. The supernatant was decanted and another 100ul of washing buffer was added followed by incubation at 52°C for 10 minutes. The mixture was centrifuged at 2000g for 10 minutes and the supernatant was decanted.
Step 4: Signal production 70 ul of washing buffer (1x Phosphate Buffer Saline, 0.01% Tween 20) containing X1/500 Streptavidin, R-phycoerythrin conjugate (Molecular Probes, Cat. S866) was added and mixed by repeat pipetting. The mixture was incubated at 52°C for 5 minutes.
Step 5: Signal measurement
The fluorescent signal was read by Luminex 100.
Results
By conventional gel electrophoresis method
Amplicons created by the above methylation specific primers were dissolved by agarose electrophoresis and ethidium bromide (Figure 3). Bisulfite-modified methylated DNA diluted in unmethylated DNA (lanes from left to right): 100:0, 10:90, 1:99, 0.1: 99.9, 0.01: 99.99 and 0.001:99.999. NTU is Non template control. M denotes the marker.
By bead-array platform according to the present invention
Amplicons created by the above methylation specific primers were assayed by Luminex(R) 100. The results are shown in Figure 4.
Figures 6 to 8 shows the sensitivity and specificity of Beads-MSP in low and high stringent conditions in a methylation specific PCR using p16.
Conclusion
The detection of DNA methylation using bead-array platform is more specific robust than the conventional agarose electrophoresis and ethidium bromide.
Example 2
Met-DNA detection using bead-array platform DNA is treated with bisulfite as discussed in Example 1.
Multiplex PCR were carried out and the methylation specific primers and probes for subsequent hybridisation were selected:
A. p16 p16 Primer set
Upper: 5'-(BiOtJn)-TATTAGAGGGTGGGGCGGATC-3' Lower: 5'-GAACCGCGACCGTAACCAA-3' p16 Probes
For 5'-C-C-3' case (2 methylations positive case)= p16 Probe2++ GG : 5'-H2NC6- tctcccctctccGcaaccGcc -3'
For 5-C-T-3' case (1 methylation positive case) = p16 Probe2++ AG : 5-H2NC6- tctcccctctccAcaaccGcc -3'
For 5T-C-3 case (1 methylation positive case) = p16 Probe2++ GA : 5'-H2NC6- tctcccctctccGcaaccAcc -3'
For 5T-T-3 case (methylation negative case) = p16 Probe2++ AA : 5-H2N-C6- tctccccctctccAcaaccAcc -3' H2N denotes amino modifier for cross linking to the bead. C6 denotes carbon spacer -
CCCCCC- between probe and bead.
B. RUNX3 RUNX3 Primer set
Upper: 5'-(BiOtJn)-GGGATAGTTACGAGGGGCGGTCGTAC-3'
Lower: 5'- GACCGACGCGAACGCCTCCT -3'
RUNX3 Probes
GG: 5'-H2N-C6-ctacccGactaatcccGcatc -3'
GA: 5'-H2N-C6-ctacccGactaatcccAcatc -3'
AG: 5'-H2N-C6-ctacccAactaatcccGcatc -3'
AA: 5'-H2N-C6-ctacccAactaatcccAcatc -3'
C. RASSF1A Set 1 (Upper primer is biotinylated)
RASSF1A Primer set
Upper: 5'-(Biotin)-TTTGCGAGAGCGCGTTTAG-3'
Lower: 5'-TAACAAACGCGAACCGAAC-3'
RASSF1A Capturing Probes
GG: 5'-H2N-C6-accccGacttcaacGcctcc -3' (Bead #17)
AG: 5'-H2N-C6-accccAacttcaacGcctcc -3' (Bead #18)
GA: 5'-H2N-C6-accccGacttcaacAcctcc -3' (Bead #19)
AA: 5'-H2N-C6-accccAacttcaacAcctccc -3' (Bead #20)
Set 2 (Lower primer is biotinylated)
RASSF1A Primer set
Upper: 5'-TTTGCGAGAGCGCGTTTAG-3'
Lower 5'-(Biotin)-TAACAAACGCGAACCGAAC-3'
RASSF1A Capturing Probes
RASSF1A S-Probe1CC: 5'-H2N-C6-gggaggCgttgaagtCggggt-3'(Bead #17)
RASSF1A S-Probe1CT: 5'-H2N-C6-gggaggCgttgaagtTggggt-3' (Bead #18)
RASSF1A S-Probe1TC: 5'-H2N-C6-gggaggTgttgaagtCggggt-3' Bead #19)
RASSF1A S-Probe1TT: 5'-H2N-C6-gggaggTgttgaagtTggggt-3' (Bead #20)
D. E-cadherin
Set 1 (Upper primer is biotinylated)
E-cadherin Primer set
Upper: 5'-(Biotin)-AGGTTAGAGGGTTATCGCGTTTATGC-3' or 5'-(BIoHn)-
AGGTTAGAGGGTTATCGCGTTTAT-3'
Lower: 5'-TACTTTACAATTCCGACGCCACT-3'
E-cadherin Capturing Probes
GG: 5'-H2N-C6-accccGaaaacaccGccccc -3' (Bead #73)
AG: 5'-H2N-C6-accccAaaaacaccGccccc -3' (Bead #74)
GA: 5'-H2N-C6-accccGaaaacaccAccccc -3' (Bead #75) AA: 5'-H2N-C6-accccAaaaacaccAccccc -3' (Bead #76)
Set 2 (Lower primer is biotinylated)
E-cadherin Primer set
Upper: 5'- AGGTTAGAGGGTTATCGCGTTTATGC-3' or 5'-(Biotin)- AGGTTAGAGGGTTATCGCGTTTAT-3' Lower: 5'-(Biotin)-TACTTTACAATTCCGACGCCACT-3' E-cadherin Capturing Probes
Ecad S-Probe 1CC: 5'-H2N-C6-gggCggtgttttCggggttt-3' (Bead #73) Ecad S-Probe 1CT: 5'-H2N-C6-gggCggtgttttTggggttt-3' (Bead #74) Ecad S-Probe 1TC: 5'-H2N-C6-gggTggtgttttCggggttt-3' (Bead #75) Ecad S-Probe 1 TT: 5'-H2N-C6-gggTggtgttttTggggttt-3' (Bead #76)
E. hMLH1 hMLH1 Primer set
Upper: 5'-(Biotin)-ATTAATAGGAAGAGCGGATAGC-3' Lower: 5'-CCTTCAACTATAACTTACGCCATC-3'
hMLH1 Probes
GG: 5'-H2N-C6-taaaacGactactacccGctacc -3' AG: 5'-H2N-C6-taaaacAactactacccGctacc -3' GA: 5'-H2N-C6-taaaacGactactacccActacc -3' AA: 5'-H2N-C6-taaaacAactactacccActacc -3'
F. GAPDH, human (This is for DNA control.) hGAPDH Primer set
Upper: 5'-Biotin-GTTTTGGGGAGGTAATTAGGAT-3' Lower: 5'-CTCACCCCAACCTTCTCCATAATA-3' hGAPDH probe
5'-H2N-C6-ccatatacccaaaaaaaccacacc-3' (only 1 probe) Multiplex PCR conditions
Multiplex PCR was performed using the QIAGEN Multiplex PCR kit (catalog no. 206143). 5ul of DNA, RUNX3 upper primer and lower primer at a final concentration of 0.1 uM and 0.05uM, p16 upper and lower primer at a final concentration of 0.1 uM and 0.1uM, RASSF1A upper and lower primer at a final concentration of 0.2uM and 0.1uM, E-cadherin upper and lower primer at a final concentration of 0.2uM and 0.1 uM, hMLH1 upper and lower primer at a final concentration of 0.2uM and 0.1 uM, and GAPDH upper and lower primer at a final concentration of 0.05uM and 0.025uM in 50uI final volume, on a Stratagene Robocycler 40 (La JoIIa, California) by using the following steps: initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 45 s, annealing at 58°C for 90 s, and extension at 72°C for 90 s, and a final extension at 72°C for 10 min.
Bead coupling for the probe is described in Example 1. The hybridisation and detection techniques are also carried out as described in Example 1.
Results
The results of the multiplex assay is shown in Figure 5. Figure 9 shows graphically the results of simultaneous detection of DNA methylation in tumour suppressor genes by a bead-based flow cytometric assay.
Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.

Claims

1. A method for detecting methylation in a DNA sample, the method comprising the steps of: (a) contacting the DNA sample with an agent that chemically converts the
DNA;
(b) amplifying the chemically converted DNA using a polymerase and at least one oligonucleotide primer carrying a detectable label; and
(c) hybridizing the amplified DNA to at least one oligonucleotide probe; wherein the methylation status of the DNA sample is determined by detecting the presence or absence of the label as a result of whether the amplified DNA hybridizes to the probe.
2. The method according to claim 1 wherein the chemically converted DNA is amplified using a polymerase and a pair of oligonucleotide primers, the pair of primers comprising an upper primer and a corresponding lower primer.
3. The method according to claim 2 wherein the upper primer carries the detectable label.
4. The method according to any one of the preceding claims wherein the agent chemically converts unmethylated cytosine residues in the DNA sample to uracil.
5. The method according to any one of the preceding claims wherein the at least one oligonucleotide probe is bonded to a solid phase.
6. The method according to claim 5 wherein the solid phase is a bead.
7. The method according to claim 6 wherein the reaction mix contains a plurality of beads, each bead has different oligonucleotide probes to capture different target regions on the amplified DNA.
8. The method according to any one of claims 6 or 7 wherein each bead has a different signature.
9. The method according to claim 8 wherein the signature is a fluorescence wavelength such that each individual bead with a separate probe is distinguishable from other beads with different probes.
10. The method according to any of the preceding claims wherein the label is biotin.
11. The method according to claim 10 further comprising the step of adding streptavidin wherein detecting the presence or absence of biotin is achieved by detecting a signal resulting from the interaction between biotin and streptavidin.
12. The method according to claim 11 wherein the streptavidin is conjugated with a fluorescent label.
13. The method according to any one of the preceding claims wherein detecting the presence or absence of the label is by fluorescent flow cytometry.
14. The method according to claim 13 wherein fluorescence is detected after irradiation with two light sources the first allowing identification of the florescence signature of the bead and the specific probe indicating the possible DNA methylation state; and the second to detect hybridisation of a sample to that probe so the amount of sample in that methylation state can be calculated.
15. The method according to any one of the preceding claims wherein the amplification is carried out by means of a polymerase chain reaction.
16. The method according to any one of the preceding claims further comprising a multiplicity of oligonucleotide primers capable of simultaneously amplifying a plurality of genes of the DNA under a single set of reaction conditions in a multiplex polymerase chain reaction.
17. The method according to claim 16 wherein the genes are selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLHL
18. The method according to any one of the preceding claims wherein more than four DNA methylation states in the DNA sample are detected in a single reaction.
19. The method according to any one of the preceding claims wherein the amplified DNA is made to hybridise with a set of four probes to detect four methylation states in a single reaction.
20. The method according to claim 19 wherein the probes having the nucleotide sequences of: GG : 5'-H2NC6-tctcccctctccGcaaccGcc -3';
AG : 5'-H2NC6-tctcccctctccAcaaccGcc -3'; GA : 5'-H2NC6-tctcccctctccGcaaccAcc -3'; AA : 5'-H2N-C6-tctccccctctccAcaaccAcc -3'; GG: 5'-H2N-C6-ctacccGactaatcccGcatc -3'; GA: 5'-H2N-C6-ctacccGactaatcccAcatc -3'; AG: 5'-H2N-C6-ctacccAactaatcccGcatc -3'; AA: 5'-H2N-C6-ctacccAactaatcccAcatc -3'; GG: 5'-H2N-C6-accccGacttcaacGcctcc -3'; AG: 5'-H2N-C6-accccAacttcaacGcctcc -3'; GA: 5'-H2N-C6-accccGacttcaacAcctcc -3'; AA: 5'-H2N-C6-accccAacttcaacAcctccc -3'; GG: 5'-H2N-C6-accccGaaaacaccGccccc -3'; AG: 5'-H2N-C6-accccAaaaacaccGccccc -3'; GA: 5'-H2N-C6-accccGaaaacaccAccccc -3'; AA: 5'-H2N-C6-accccAaaaacaccAccccc -3'; GG: 5'-H2N-C6-taaaacGactactacccGctacc -3'; AG: 5'-H2N-C6-taaaacAactactacccGctacc -3'; GA: 5'-H2N-C6-taaaacGactactacccActacc -3'; AA: 5'-H2N-C6-taaaacAactactacccActacc -3'; CC: 5'-H2N-C6-gggaggCgttgaagtCggggt-3'; CT: 5'-H2N-C6-gggaggCgttgaagtTggggt-3'; TC: 5'-H2N-C6-gggaggTgttgaagtCggggt-3'; TT: 5'-H2N-C6-gggaggTgttgaagtTggggt-3'; CC: 5'-H2N-C6-gggCggtgttttCggggttt-3'; CT: 5'-H2N-C6-gggCggtgttttTggggttt-3'; TC: 5'-H2N-C6-gggTggtgttttCggggttt-3'; TT: 5'-H2N-C6-gggTggtgtttfrggggttt-3'; or 5'-H2N-C6-ccatatacccaaaaaaaccacacc-3'.
21. The method according to any one of the preceding claims wherein the agent is a bisulfite solution.
22. The method according to any one of the preceding claims wherein the polymerase is a heat-resistant DNA polymerase.
23. The method according to any one of the preceding claims wherein the methylation status of genes selected from the group RUNX3, p16, RASSF1A, E- cadherin and hMLH1 in the DNA sample is determined in a single run.
24. A primer set comprising at least two primers capable of amplifying a portion of the sequence of p16.
25. The primer set according to claim 24 having the nucleotide sequence 5'-Biotin- GTTATTAGAGGGTGGGGCGGATCG-3' / 5'- AACGCCCCCGCCTCCAACAACG-3'.
26. A primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the p16 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
27. A primer set comprising at least two primers capable of amplifying a portion of the sequence of RUNX3 gene.
28. The primer set according to claim 27 having the nucleotide sequence 5'-(Biotin)- GGGATAGTTACGAGGGGCGGTCGTAC-3' / 5'- GACCGACGCGAACGCCTCCT -3'.
29. A primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the RUNX3 gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
30. A primer set comprising at least two primers capable of amplifying the promoter sequence of RASSF1 A gene.
31. The primer set according to claim 30 having the nucleotide sequence 5'-(Biotin)- TTTGCGAGAGCGCGTTTAG-3' / 5'-TAACAAACGCGAACCGAAC-3' or 5'- TTTGCGAGAGCGCGTTTAG-3' / 5'-(Biotin)-TAACAAACGCGAACCGAAC-3\
32. A primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the RASSF1A gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
33. A primer set comprising at least two primers capable of amplifying the promoter sequence of E-cadherin gene.
34. The primer set according to claim 33 having the nucleotide sequence 5'-(Biotin)- AGGTTAGAGGGTTATCGCGTTTAT-3' / 5'-TACTTTACAATTCCGACGCCACT- 3' or, 5'-AGGTTAGAGGGTTATCGCGTTTAT-3' / 5'-(Biotin)- TACTTTACAATTCCGACGCCACT-3' or, 5'-(Biotin)- AGGTTAGAGGGTTATCGCGTTTATGC-3' / 5'- TACTTTACAATTCCGACGCCACT-3' or, 5'- AGGTTAGAGGGTTATCGCGTTTATGC-3' / 5'-(Biotin)- TACTTTACAATTCCGACGCCACT-3' .
35. A primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the E-cadherin gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
36. A primer set comprising at least two primers capable of amplifying the promoter sequence of hMLH1 gene.
37. The primer set according to claim 36 having the nucleotide sequence 5'-(Biotin)- ATTAATAGGAAGAGCGGATAGC-3' / 5'-CCTTCAACTATAACTTACGCCATC- 3'.
38. A primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of a portion of the hMLHM gene under a single set of reaction conditions in a multiplex polymerase chain reaction.
39. A primer set comprising a multiplicity of primers capable of simultaneously amplifying a plurality of genes selected from RUNX3, p16, RASSF1A, E-cadherin and hMLH1 under a single set of reaction conditions in a multiplex polymerase chain reaction.
40. An oligonucleotide probe having a nucleotide sequence that hybridizes under stringent conditions to the complement of DNA sequences of RUNX3 and/or p16 and/or RASSF1A and/or E-cadherin and/or hMLHL
41. Oligonucleotide probes having the nucleotide sequences of: GG : 5'-H2NC6-tctcccctctccGcaaccGcc -3';
AG : 5'-H2NC6-tctcccctctccAcaaccGcc -3'; GA : 5'-H2NC6-tctcccctctccGcaaccAcc -3'; AA : 5'-H2N-C6-tctccccctctccAcaaccAcc -3;' GG: 5'-H2N-C6-ctacccGactaatcccGcatc -3'; GA: 5'-H2N-C6-ctacccGactaatcccAcatc -3'; AG: 5'-H2N-C6-ctacccAactaatcccGcatc -3'; AA: 5'-H2N-C6-ctacccAactaatcccAcatc -3'; GG: 5'-H2N-C6-accccGacttcaacGcctcc -3'; AG: 5'-H2N-C6-accccAacttcaacGcctcc -3'; GA: 5'-H2N-C6-accccGacttcaacAcctcc -3'; AA: 5'-H2N-C6-accccAacttcaacAcctccc -3'; GG: 5'-H2N-C6-accccGaaaacaccGccccc -3'; AG: 5'-H2N-C6-accccAaaaacaccGccccc -3'; GA: 5'-H2N-C6-accccGaaaacaccAccccc -3'; AA: 5'-H2N-C6-accccAaaaacaccAccccc -3'; GG: 5'-H2N-C6-taaaacGactactacccGctacc -3'; AG: 5'-H2N-C6-taaaacAactactacccGctacc -3'; GA: 5'-H2N-C6-taaaacGactactacccActacc -3'; AA: 5'-H2N-C6-taaaacAactactacccActacc -3'; CC: 5'-H2N-C6-gggaggCgttgaagtCggggt-3'; CT: 5'-H2N-C6-gggaggCgttgaagtTggggt-3'; TC: 5'-H2N-C6-gggaggTgttgaagtCggggt-3'; TT: 5'-H2N-C6-gggaggTgttgaagtTggggt-3';
CC: 5'-H2N-C6-gggCggtgttttCggggttt-3'; CT: 5'-H2N-C6-gggCggtgttttTggggttt-3';
TC: 5'-H2N-C6-gggTggtgttttCggggttt-3'; TT: 5'-H2N-C6-gggTggtgttttTggggttt-3'; or 5'-H2N-C6-ccatatacccaaaaaaaccacacc-3'.
42. A method of aiding assessment of a patient's risk of developing cancer, or progression of cancer, the method comprising the step of obtaining a sample containing nucleic acid from a patient and carrying out the method according any one of claims 1 to 23.
43. A method of diagnosing or assessing a patient's risk of developing cancer or other diseases by detecting DNA methylation in genes selected from the group RUNX3, p16, RASSF1A, E-cadherin and hMLH1, by carrying out the method according any one of claims 1 to 23.
44. A kit of parts comprising:
(a) one or more primers according to any one of the claims 24 to 39, wherein the primer or primers carry a detectable label;
(b) one or more probes according to any one of the claims 40 or 41 ;
(c) means for detecting the label; and (d) directions for performing the method according to any one of claims 1 to
23.
45. The kit according to claim 44 wherein the label is biotin.
46. The kit according to claim 45 further comprising streptavidin wherein detecting the presence or absence of the label biotin is determined by detecting a signal resulting from the interaction between biotin and streptavidin.
47. The kit according to claim 46 wherein the streptavidin is conjugated with a fluorescent label.
48. The kit according to any one of claims 44 to 47 wherein the means for detecting the label is fluorescent flow cytometry.
PCT/SG2006/000271 2005-09-15 2006-09-15 Method for detecting dna methylation WO2007032748A1 (en)

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